Received: July 16, 2018. Accepted: October 30, 2018. Pre-published: October 31, 2018.
©2019 Ferrata Storti Foundation
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Correspondence:
MARIA ROSARIA SAPIENZAmariarosaria.sapienza@gmail.com
Haematologica
2019
Volume 104(4):729-737
doi:10.3324/haematol.2018.202093
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/4/729
Ferrata Storti Foundation
B
lastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and
aggressive hematologic malignancy for which there is still no
effec-tive therapy. In order to identify genetic alterations useful for a new
treatment design, we used whole-exome sequencing to analyze 14
BPDCN patients and the patient-derived CAL-1 cell line. The functional
enrichment analysis of mutational data reported the epigenetic
regulato-ry program to be the most significantly undermined (P<0.0001). In
par-ticular, twenty-five epigenetic modifiers were found mutated (e.g.
ASXL1, TET2, SUZ12, ARID1A, PHF2, CHD8); ASXL1 was the most
fre-quently affected (28.6% of cases). To evaluate the impact of the
identi-fied epigenetic mutations at the gene-expression and Histone H3 lysine
27 trimethylation/acetylation levels, we performed additional RNA and
pathology tissue-chromatin immunoprecipitation sequencing
experi-ments. The patients displayed enrichment in gene signatures regulated
by methylation and modifiable by decitabine administration, shared
common H3K27-acetylated regions, and had a set of cell-cycle genes
aberrantly up-regulated and marked by promoter acetylation.
Collectively, the integration of sequencing data showed the potential of
a therapy based on epigenetic agents. Through the adoption of a
preclin-ical BPDCN mouse model, established by CAL-1 cell line xenografting,
we demonstrated the efficacy of the combination of the epigenetic drugs
5’-azacytidine and decitabine in controlling disease progression in vivo.
Blastic plasmacytoid dendritic cell
neoplasm: genomics mark epigenetic
dysregulation as a primary therapeutic target
Maria Rosaria Sapienza,1*Francesco Abate,2,3*Federica Melle,4Stefania Orecchioni,5Fabio Fuligni,6Maryam Etebari,1Valentina Tabanelli,4
Maria Antonella Laginestra,1Alessandro Pileri,7,8Giovanna Motta,4
Maura Rossi,1Claudio Agostinelli,1Elena Sabattini,1Nicola Pimpinelli,8
Mauro Truni,9Brunangelo Falini,10Lorenzo Cerroni,11Giovanna Talarico,5
Rossana Piccioni,12Stefano Amente,13Valentina Indio,14
Giuseppe Tarantino,14Francesco Brundu,2Marco Paulli,15Emilio Berti,16
Fabio Facchetti,17Gaetano Ivan Dellino,12,18 Francesco Bertolini,5
Claudio Tripodo,19*Raul Rabadan2,3* and Stefano A. Pileri4ǂ*
1Hematopathology Unit, Department of Experimental, Diagnostic, and Specialty Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Italy; 2Department of Systems Biology, Columbia University College of Physicians and Surgeons, New York, NY, USA; 3Department of Biomedical Informatics, Columbia University College of Physicians and Surgeons, New York, NY, USA; 4Division of Haematopathology, IEO European Institute of Oncology IRCCS, Milan, Italy; 5Laboratory of Hematology-Oncology, IEO European Institute of Oncology IRCCS, Milan, Italy; 6Department of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada: 7Dermatology Unit, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Italy; 8Division of Dermatology, Department of Surgery and Translational Medicine, University of Florence, Italy; 9Pathological Anatomy Histology & Cytogenetics, Niguarda Cancer Center, Niguarda-Ca' Granda Hospital, Milan, Italy; 10Institute of Hematology and Center for Hemato-Oncology Research (CREO), University and Hospital of Perugia, Italy; 11Universitätsklinik für Dermatologie und Venerologie,
LKH-Universitatsklinikum Graz, Austria; 12Department of Experimental Oncology, European Institute of Oncology, Milan, Italy; 13Department of Molecular Medicine and Medical Biotechnologies, University of Naples ‘Federico II’, Italy; 14"Giorgio Prodi" Cancer Research Center, University of Bologna, Italy; 15Unit of Anatomic Pathology, Department of Molecular Medicine, University of Pavia and Fondazione IRCCS San Matteo Policlinic, Pavia, Italy; 16Department of Dermatology, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinic and Milan University, Milan, Italy; 17Pathology Section, Department of Molecular and Translational Medicine, University of Brescia, Italy; 18Department of Oncology and Hemato-Oncology, University of Milan, Italy and 19Tumor Immunology Unit, Department of Health Science, Human Pathology Section, University of Palermo School of Medicine, Italy
*MRS, FA, CT, RR and SAP contributed equally to this work. ǂAlma Mater Professor, Bologna University
Introduction
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare malignancy derived from precursors of plasma-cytoid dendritic cells.1-4 It has no clear racial or ethnic
predisposition and more often affects elderly males (male/female ratio 3.3:1; mean/median age at diagnosis: 61-67 years). BPDCN patients usually respond to first-line chemotherapy, but despite this they almost invari-ably relapse and display a dismal prognosis with a medi-an overall survival (OS) rmedi-anging from 10 to 19 months.2
No standardized therapeutic approach has so far been established for BPDCN, even if hematopoietic stem cell transplantation has been shown to achieve remission in selected patients.5-6Therefore, the development of
effec-tive treatments still represents an unmet need.7 The
pathobiology of BPDCN is poorly understood and the number of reports exploring its molecular features is still limited.8-21 Recent advances in the understanding of the
BPDCN molecular landscape have paved the way for novel treatment approaches based on the inhibition of the BCL2 protein,22 the activation of the cholesterol
efflux,23the repression of the Bromodomain-containing
protein 4 (BRD4),24 and binding to the interleukin-3
receptor (IL3R).25 All these potential therapeutic options
(which are worthy of further evaluation) have mainly emerged from the analysis of the BPDCN transcriptome or from its antigenic repertoire. The genomic landscape of BPDCN has not been well investigated, and only a few studies have explored the mutational events occur-ring in BPDCN, mainly through targeted sequencing approaches.14,16,19,20
Unfortunately, these have not offered any novel prospects of treatment options.
In this study, we performed whole-exome sequencing (WES) of 14 BPDCN samples and of the BPDCN-derived CAL-1 cell line to look for specific BPDCN genetic vulner-abilities that may support the design of new therapeutic strategies. The WES mutational findings were comple-mented by copy number variant (CNV) analysis, RNA and pathology tissue-chromatin immunoprecipitation (PAT-ChIP) sequencing results. The integration of data allowed us to identify a successful combinatorial therapy based on epigenetic drugs able to control disease progression in a rapidly progressive BPDCN xenograft model.
Methods
Blastic plasmacytoid dendritic cell neoplasm samples
We collected 14 BPDCN cryopreserved cutaneous biop-sies at diagnosis, 9 matched saliva samples and the BPDCN patient-derived cell line, CAL-1. The pathological cases were evaluated as previously described17anddiag-nosed by experienced hematopathologists (CA, EB, FF, LC, MP, ES, CT, MT, and SAP) according to World Health Organization diagnostic criteria.2 Informed consent was
obtained from each patient in accordance with the Ethical Review Board of the Department of Experimental, Diagnostic, and Specialty Medicine of the University of Bologna, Italy, and the Declaration of Helsinki. DNA was extracted as reported in the Online Supplementary
Appendix. The main clinical, immunohistochemical and
cytogenetic features of the BPDCN patients are shown in
Online Supplementary Tables S1 and S2.
Whole-exome sequencing analysis
We performed paired-end sequencing of matched tumor/normal DNA samples (9 cases), tumor only DNA samples (5 cases), and the CAL-1 cell line (Online
Supplementary Table S3) using the TruSeq Exome Kit and
Nextera Rapid Capture Exome kit (Illumina). Further details are available in the Online Supplementary Appendix.
Sanger sequencing
We used Sanger sequencing to analyze two candidate nonsense somatic mutations of SUZ12 and ASXL1 occurring in 2 patients, respectively, as described in the Online
Supplementary Appendix.
Targeted sequencing
We performed MiSeq targeted sequencing (Illumina) of the 14 BPDCN tumor patients, 7 normal matched saliva samples and the CAL-1 cell line, already analyzed by WES. More bioinformatics details are provided in the Online
Supplementary Appendix and Online Supplementary Tables S4 and S5.
RNA sequencing analysis
Five BPDCN cases studied by WES and targeted ing had sufficient material for RNA extraction and sequenc-ing; these samples represented the RNA sequencing (RNA-seq) discovery set. We also collected an additional 4 BPDCN cryopreserved cutaneous biopsies, sufficient only for RNA sequencing experiments, used as an RNA-seq extension set. RNA of 4 normal plasmacytoid dendritic cell (pDCs) sam-ples was purchased from AllCells (Alameda, CA, US) and used for comparison. For details, see Online Supplementary
Table S6 and the Online Supplementary Appendix.
Pathology tissue-chromatin immunoprecipitation
sequencing
The BPDCN_25 and BPDCN_37 patients were provided with one skin biopsy: half was cryopreserved and used for WES, targeted and RNA sequencing analysis, and the other half was fixed in formalin, included in paraffin and used for pathology tissue-chromatin immunoprecipitation (PAT-ChIP) sequencing analysis. PAT-ChIP experiments were per-formed as in Fanelli et al.26 Further details are available in the Online Supplementary Appendix.
CAL-1 cell line
CAL-1, a BPDCN cell line27 was cultured as reported
pre-viously.18 The CAL-1 gene expression profile of a previous
study was used17 (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE62014).
Mouse model and in vivo treatments
Experiments were carried out on 6-8-week old non-obese diabetic severe combined immunodeficient NOD/SCID interleukin-2 receptor g (IL-2Rg)–null (NSG) mice, as previously reported.13 All animal experiments
were carried out in accordance with the Italian laws in force (Legislative Decree 26/14 and subsequent amend-ments) and institutional guidelines. All in vivo studies were ratified by the Italian Ministry of Health. For induction of BPDCN in mice, 5000 CAL-1 cells were injected intra-venously (i.v.) through the lateral tail vein in non-irradiat-ed mice. Engraftnon-irradiat-ed mice were then treatnon-irradiat-ed with borte-zomib, 5’-azacytidine, decitabine and romidepsin, as detailed in the Online Supplementary Appendix.
Figure 1. The genomic characterization of blastic plasmacy-toid dendritic cell neoplasm (BPDCN). (A) Circos plot graphi-cal representation of the functional analysis performed on 54 genes recurrently mutated and/or affected by nonsense and frameshift single nucleotide variants (SNVs) in BPDCN whole-exome sequencing (WES) samples. The four biological processes most significantly enriched are reported in the counterclockwise order from the highest to the lowest
P-value: the gamma-aminobutyric acid (GABA) secretion (in
violet), the Rac signaling (in red), the hematopoietic stem cell homeostasis (in light blue) and the epigenetic process (in green). The genes are colored according to their belonging to one or more of the biological processes represented. Genes not involved are in gray. (B) Overview of the TET2 and ASXL1 mutations identified. Structure of ASXL1 protein with C-termi-nal plant homeodomain catalytic region and structure of TET2 protein showing the catalytic core region: the cysteine-rich (Cys) and double-stranded β-helix (DSBH) domains. Empty circles: somatic SNVs. + : frameshift SNV; *nonsense SNV. (C) Heatmap representation of SNVs in BPDCN WES samples and its distribution among selected pathways com-monly mutated in myeloid disorders. The SNVs, the affected genes and the related pathways are reported in rows, while, the BPDCN samples are in columns.
A
C
B
Results
Whole-exome sequencing reveals the epigenetic
pro-gram dysregulation as the main theme of the blastic
plasmacytoid dendritic cell neoplasm mutational
landscape
We collected 14 BPDCN cases with a mean age of 56 years at diagnosis (range 9-89 years), a male-to-female
ratio of 10:4, and the classical BPDCN presentation (Online
Supplementary Tables S1 and S2).1 The enrolled patients
underwent different treatment regimens and 78.5% (11 out of 14) died of the disease 6.3-76 months after the diag-nosis or were lost at follow up. Most patients who under-went autologous and/or allogeneic hematopoietic stem cell transplantation experienced a prolonged survival.
Figure 2. The transcriptome and H3K27 trymethylation/acetylation profiling of blastic plasmacytoid dendritic cell neoplasm (BPDCN).(A) Unsupervised hierarchi-cal clustering performed on 5 BPDCN samples and 4 plasmacytoid dendritic cell (pDCs) samples according to the expression level of the RNA sequencing data. In the heat-map each row represents a gene and each column a sample. The color scale exemplifies the relative expression level of a gene across all samples: (red) represented genes with an expression level above the mean; (blue) the genes with an expression level lower than the mean. Tumors (BPDCNs) and controls (pDCs) cluster in two distinct groups. (B) Gene Set Enrichment Analysis (GSEA) plot illustrating the enrichment of the KDM5B and PRMT5 gene signatures in BPDCN patients reported in literature34-36as well as the enrichment of a set of genes, described by Missiaglia et al.37as responsive to hypomethylating treatment, namely decitabine.
Normalized enrichment score (NES) ≥ 2; false discovery rate (FDR) q-value false discovery rate ≤0.0001. (C) Visualization of anti-H3K27ac and anti-H3K27me3
nor-malized pathology tissue-chromatin immunoprecipitation (PAT-ChIP) sequencing profiles in the UCSC Genome Browser showing genomic regions from patient BPDCN_25 and BPDCN_37. (Red boxes) Exemplificative regions displaying a similar level of anti-H3K27ac in both patients. (Black solid rectangles) Genes in corre-spondence of the anti-H3K27ac peaks. (D) The cases BPDCN_25 and BPDCN_37 share common H3k27ac regions. (E) Histogram representation of the top 10 sig-nificant biological processes emerged by Gene Ontology (GO) analysis of 86 up-regulated genes marked by H3K27ac in their promoters. GO categories are shown in x-axis and the fold enrichment values of observed versus expected genes are reported in the y-axis (FDR q-value <0.001).
C
Figure 3. The efficacy of epigenetic agents in a preclinical blastic plasmacytoid dendritic cell neoplasm (BPDCN) mouse model.(A) Pharmacodynamic assessment
of the percentage of human CD56+CD38+cells in the peripheral blood (PB), bone marrow (BM) of the femur and spine, spleen, and liver of a representative
vehicle-treated BPDCN mouse model, 39 days after CAL-1 injection. The cytofluorometric assays shows the tumor dissemination in all the tissues analyzed. (B) Hematoxylin & eosin (H&E) staining of BM and spleen samples collected in a representative vehicle-treated NSG mouse 39 days after CAL-1 injection (H&E; x400; Olympus DP2-SAL). The histological assay shows a marked dissemination of blast elements. The immunohistochemistry detection of the CD303 (BDCA-2) antigen, in the murine BM, indicates the presence of specific BPDCN blasts cells (Immmuno-alkaline phosphatase; Gill’s hematoxylin nuclear counterstaining; x400; Olympus DP2-SAL). These results further confirmed the effective engraftment of CAL-1 cell line. (C) Graphical representation of the treatment schedules observed in a BPDCN mouse model. Each treatment is represented by a single color or by a combination of colors and was administered for four weeks as follows: 5’-azacytidine 5 mg/kg 5 doses at 2-day intervals (green), decitabine 2.5 mg/kg 3 doses at 2-day intervals (light brown), romidepsin 0.5 mg/kg every day (violet), bortezomib 0.5 mg/kg two times weekly (fuchsia). The same doses were also administered in various combinations. (D) Kaplan-Meier curves comparing overall survival of BPDCN mice models respec-tively treated with vehicle or the above reported treatments. Each treatment is summarized by a box colored as described above. *Indicates that the combination of decitabine and 5’-azacytidine was the most effective in prolonging mice survival. Curves were compared by log-rank test, n=5 mice/treatment arm. (E) Pharmacodynamic assessment of spleen size in 4 representative NSG mice CAL-1 injected after 39 days of treatment with vehicle (mouse Control), Decitabine (mouse Deci), 5’-azacytidine (mouse Aza), and 5’-azacytidine plus decitabine (mouse Deci+Aza) according to the dosing schedule reported above. ns: not significant.
A
B
C
D
BPDCN patient-derived CAL-1 cell line and identified 1302 non-synonymous single nucleotide variants (SNVs) represented predominantly by missense SNVs (n=1251), nonsense SNVs (n=47), and frameshift insertions/dele-tions (n=7). To verify the robustness of the WES analysis, 2 randomly-selected variants occurring in the ASXL1 and
SUZ12 genes, respectively, were validated by Sanger
sequencing (Online Supplementary Figure S1). To extend the validation to a higher number of samples and mutations, a targeted sequencing approach was adopted: 21 SNVs were interrogated by MiSeq Illumina technology in the same BPDCN cases analyzed by WES and a concordance of 95.2% (20 out of 21 SNVs) was achieved, underlining a high consistency of data (Online Supplementary Table S7). The 1302 non-synonymous SNVs detected by WES affect-ed 1166 genes, all but 7 known to be relataffect-ed with patho-logical conditions and reported as mutated in the Catalogue of Somatic Mutations in Cancer (COSMIC_v66).
To identify the biological processes that were most altered by the mutational events, we performed a func-tional enrichment analysis of the 9 genes that were recur-rently mutated (≥ 3 samples) and the 45 genes impacted by deleterious (nonsense or frameshift) SNVs. Among the top 10 significantly enriched biological processes, the epigenetic program was the most represented (P=0.0001), followed by hematopoietic stem cell home-ostasis, Rac signaling and gamma-aminobutyric acid (GABA) secretion (Figure 1A and Online Supplementary
Table S8). The ASXL1 gene proved to be the most
fre-quently mutated (28%, 4 out of 14 samples), followed by
TET2 (21%, 3 out of 14 samples); both genes displayed
mainly nonsense or frameshift SNVs located within or upstream of the catalytic domain of the proteins, poten-tially leading to their functional disruption (Figure 1B and
Online Supplementary Table S9). We consulted the Gene
Ontology database28to identify among the 1166 BPDCN
mutated genes those implicated in the epigenetic regula-tion. We found 25 mutated epigenetic modifier genes controlling chromatin accessibility (ARID1a, CHD8,
SMARCA1), DNA methylation (TET2, IDH2), or histone
post-transcriptional modifications [methylation (ASXL1,
SUZ12, MLL family), demethylation (KDM4D),
acetyla-tion (EP300, EP400), ubiquitinaacetyla-tion (PHC1, PHC2), dephosphorylation (EYA2), and exchange (SRCAP)]. Of note, 12 out of 14 BPDCN samples (86%) harbored at least one of the 25 epigenetic regulator genes mutated, and specifically 8 out of 14 patients (57.14%) presented a deleterious lesion (nonsense/frameshift SNV) (Online
Supplementary Table S10). Many SNVs clustered in the
histone methylation pathway, specifically in genes belonging to the Polycomb-repressive complex 2 (ASXL1, ASXL3, SUZ12) and in histone methyltrans-ferases (ASHL1, SETMAR, MLL), possibly compromising the integrity of the methylation program. Besides genetic lesions targeting epigenetic regulators, we also detected mutations potentially affecting molecular programs com-monly deregulated in myeloid malignancies, such as RAS signaling29 (hot-spot SNVs on KRAS or NRAS,
alterna-tively), DNA repair/cycle progression30(SNVs on BRCA1,
ATM, ATR, and RAD52), Wnt-signaling31 (SNVs on
WNT3, WNT7B, WNT10 and BCL9L), cell growth32 (SNVs
on RUNX2, MAPK1), and splicing machinery33(an SNV
on ZRSR2) (Figure 1C).
Whole-exome sequencing data were also used for
cytogenetic CNV analysis, which highlighted extensive losses along the chromosome 9 and the associated dele-tion of the tumor suppressor CDKN2A gene in 8 out of 14 BPDCN samples (57%) (Online Supplementary Figure
S2), as already reported in the literature.12,15,20In addition,
CNV analysis showed that deletions affected six of the nine genes recurrently mutated; deletions were always mutually exclusive with mutations (Online Supplementary
Figure S3). However, no significant correlation was found
between genetic lesions and the clinical data.
Blastic plasmacytoid dendritic cell neoplasm
tran-scriptome profiling confirms the dysregulation of
epi-genetic programs
Genetic lesions in key epigenetic modifier genes and in related regulatory networks can induce profound pertur-bations in the transcriptional homeostasis of the cell. To further substantiate the impact of mutations affecting the chromatin remodeling pathway in BPDCN, we per-formed RNA sequencing of 5 BPDCNs, considered as the discovery set, already studied by WES and MiSeq target-ed sequencing. We compartarget-ed the patients' transcrip-tomes with those of 4 normal plasmacytoid dendritic cell (pDC) samples isolated from the peripheral blood of healthy individuals and used as controls. BPDCN tumor samples and pDCs segregated separately according to their gene expression profiles (Figure 2A). Two thousand and thirty-four genes (2034) were significantly deregulat-ed among patients, and approximately half of them were up-regulated (46%) in the BPDCN setting. Gene set enrichment analysis (GSEA) reported the significant deregulation of two genetic signatures involved in the methylation process, driven by the KDM5B34 histone demethylase and PRMT535 methyltransferase-associat-ed genes, respectively. Of note, GSEA also detectmethyltransferase-associat-ed the significant enrichment of a set of genes associated with the response to a DNA demethylating agent,36 namely
decitabine (Figure 2B). The GSEA results [normalized enrichment score (NES) ≥2; false discovery rate (FDR) q-value ≤0.0001] were then validated in an extension set of 4 BPDCN samples and in a CAL-1 cell line (Online
Supplementary Figures S4 and S5).
Genome-wide ChIP-sequencing substantiates
epigenetic dysregulation of cell cycle program
in blastic plasmacytoid dendritic cell neoplasms
To investigate if the transcriptional deregulation of BPDCNs could be linked to specific epigenetic features, we analyzed the histone acetylation/methylation pro-files of 2 selected BPDCN patients (BPDCN_25 and BPDCN_37). The trimethylation at lysine 27 of histone 3 (H3K27me3) is closely associated with inactive gene pro-moters, while its acetylation (H3K27ac) closely correlates with gene activation, the two epigenetic modifications being mutually exclusive. Given this, we analyzed the genome-wide distribution of trimethylation and acetyla-tion profiles of H3K27 in BPDCN cases. The analysis of PAT-ChIP sequencing data demonstrated that the 2 patients converged on the same pattern of histone acety-lation, sharing as much as 43.6% of the acetylated pro-moters (Figure 2C and D). PAT-ChIP sequencing results were then integrated into the RNA sequencing data lead-ing to the identification of a signature of 86 genes marked by promoter acetylation and significantly over-expressed in the BPDCN RNA sequencing sets. Gene
Ontology analysis of the 86 selected genes highlighted the enrichment in biological processes involved in cell cycle progression (FDR q-value <0.001) (Figure 2E and
Online Supplementary Table S11).
In vivo blastic plasmacytoid dendritic cell neoplasm
modeling demonstrates combined epigenetic therapy
as effective in controlling disease progression
The integration of results obtained from WES, RNA sequencing and PAT-ChIP-sequencing experiments clear-ly pointed to a fundamental role for epigenetic dysregu-lation in BPDCN and allowed us to hypothesize that this malignancy could display susceptibility to drugs active on the epigenetic regulation. Following the demonstra-tion that the CAL-1 cell line, like primary BPDCN sam-ples, had mutations clustering in chromatin remodeling pathway (Figure 1C) and enrichment in the same epige-netic programs (Online Supplementary Figure S5), we developed an in vivo CAL-1 xenograft BPDCN-like model to explore the effects of treatments targeting the acetyla-tion, methylaacetyla-tion, and also the NF-kB pathways, accord-ing to previous results.17,18 To this end, we focused on
four different
FDA-approved compounds: 5’-azacytidine, decitabine, romidepsin and bortezomib.
NSG mice intravenously injected with 5x103 CAL-1
cells rapidly developed a systemic BPDCN-like progres-sive disease, which was defined by the flow cytometry identification of human CD56+CD38+malignant cells in
the peripheral blood, bone marrow, spleen and liver, as evaluated at 39 days after injection (Figure 3A). The pathological infiltration by malignant BPDCN cells in the mouse model was also confirmed at the same time point by the histopathological analysis of the bone marrow and spleen samples, which showed the presence of atyp-ical cells with blastic morphology and expressing the human CD303/BDCA2 pDC marker (Figure 3B). Xenografted mice were divided into 11 treatment groups (n=110 mice) one day after CAL-1 injection and treated with either saline or with the hypo-methylating agents 5’-azacitidine or decitabine, the proteasome inhibitor bortezomib, and the histone deacetylase inhibitor romidepsin, used as single agents or in combination, according to the treatment schedule summarized in Figure 3C. The administration of 5’-azacytidine and decitabine used as single agents significantly prolonged OS of the mice when compared with saline (median sur-vival 43.6 days vs. 32 days, P<0.01 for 5’-azacytidine; median survival 44.7 days vs. 32 days, P<0.05 for decitabine) while neither bortezomib nor romidepsin alone showed beneficial effects on disease outcome. When the same agents were associated in combined treatment experiments, three different combinations were seen to significantly prolong mouse survival: i) the association of romidepsin and decitabine (median sur-vival 42.8 days vs. 32 days, P<0.05); ii) the combination of romidepsin, decitabine, and 5’-azacytidine (median survival 41.8 days vs. 32 days, P<0.01); and iii) the asso-ciation of decitabine and 5’-azacytidine (median survival 52.8 days vs. 32 days, P<0.01), which achieved the best result in terms of survival (Figure 3D). Consistently, 5’-azacytdine and decitabine administered alone reduced the CAL-1-induced splenomegaly as evaluated at day 39 post injection and their combination proved to be even more effective (Figure 3E).
Discussion
Blastic plasmacytoid dendritic cell neoplasm is a rare myeloid malignancy with dismal prognosis and no stan-dard therapy. In the present study, we performed WES on the largest series of BPDCNs that, to the best of our knowledge, has so far been reported in the literature. Thanks to the integration of WES with RNA and PAT-ChIP sequencing, we provide new insights into BPDCN pathobiology by highlighting the dysregulation of the epi-genetic program as a hallmark of the disease and suggest possible novel therapeutic interventions.
We found BPDCN patients extensively affected by mutations of genes involved in the epigenetic regulation: 25 epigenetic modifiers were mutated in almost all BPDCN patients (13 out of 14) and the CAL-1 cell line. In more than half of the patients (8 out of 14), the mutations heralded damaging functional alterations (Figure 1C). Some of the mutated genes have already been reported in previous studies (e.g. ASXL1, RAS, ATM, ARID1A, and
IDH2), although, at times, at different rates than in our
series (see ASXL1 and TET2, which were found to be mutated in 28.6% and 21.4% of our samples vs. 32% and 36% of those of Menezes et al.19). In this respect, it should
be remembered that the aim of our study was not only to extensively explore the mutational landscape of BPDCN, but also to possibly translate molecular notions into a pre-clinical approach. In any case, thanks to the employment of a WES approach, which did not limit our investigation to a priori-selected genes, we recognized additional mutat-ed epigenetic factors that have never been describmutat-ed before but which are potentially relevant in the context of BPDCN, like PHF2 histone demethylase, that enhances the TP53-tumor suppressor activity,37 and the CHD8
Chromodomain helicase DNA-binding protein-8, that promotes the E2F-dependent transcription and cell cycle progression.38 Besides the epigenetic pathway, we also
detected mutations affecting programs common to other myeloid malignancies, such as DNA repair process,30
Wnt/β-catenin signaling,31 and the differentiation
path-way.32
Importantly, the functional enrichment analysis of WES data showed that among all genes/pathways explored the epigenetic program was the most deregulated (P<0.0001). To evaluate the impact of the identified epigenetic mutations at gene expression level, we analyzed the tran-scriptome of samples studied by WES. Among up-regulat-ed genes, GSEA revealup-regulat-ed the significant enrichment of two methylation pathways, driven respectively by the
KDM5B histone-demethylase34and by the PRMT5
argi-nine methyltransferase-5;35these two epigenetic modifiers
are reported to be over-expressed in several cancer types and also represent promising therapeutic targets.39
Blockade of the PRMT5 activity reduces cell survival in chronic myelogenous leukemia40 and inhibition of KDM5B demethylation correlates with cell growth arrest
in hepatocellular carcinoma and breast cancers.41,42 We also
identified the overexpression of one gene signature36
specifically responsive to the administration of the hypomethylating agent decitabine; a molecular finding bearing important therapeutic implications (FDR q=1.85E-5). To gain a functional insight into the epigenetic land-scape of BPDCN samples, we performed PAT-ChIP sequencing of H3K27-acetylation/trymethylation signals of 2 BPDCN patients. The trimethylation of H3K27 marks
inactive gene promoters and enhancers, while its acetyla-tion correlates with gene activaacetyla-tion.43PAT-ChIP
sequenc-ing data showed that the 2 patients converged on the same epigenetic pattern sharing approximately half of the identified H3K27-acetylated promoters. Interestingly, the common acetylated regions comprised 10 super-enhancers (SE) bound by the Bromodomain-containing protein 4 (BRD4), as described by Ceribelli et al. in a recent work on BPDCN (data not shown).24
The integration of PAT-ChIP and the RNA sequencing data highlighted a set of 86 genes involved in the cell-cycle progression aberrantly over-expressed and marked by H3K27-promoter acetylation. This finding suggests that the cell-cycle deregulation could be driven by H3K27-acetylation signals, a hypothesis meriting future ad hoc studies that could help to clarify the mechanism of prolif-eration of this largely obscure disease.
The rarity of the disease (with an incidence of 0.000045%) and its extremely aggressive behavior (OS 10-19 months) limits the number of available patients includ-ed in biological and/or clinical studies. For these reasons, not surprisingly, BPDCN is still an orphan tumor lacking a standardized and effective therapeutic approach. In the last few years, new molecular studies have opened the way to innovative target therapies (e.g. bortezomib,17,18
venetoclax,22 BET-inhibitors,24 SL-40125) being used in
clinical trials. Some of these are showing promising results, although still concerns remain regarding their safe-ty. Of note, all the treatments proposed are mainly the result of investigation into the RNA transcriptome, while the DNA features of BPDCN patients have barely been evaluated.
We therefore decided to tackle this yet incurable dis-ease by designing the first therapeutic strategy modeled on the DNA mutational status of BPDCN patients, ana-lyzed by WES. The WES mutational findings enhanced by the RNA and PAT-ChIP sequencing results clearly evi-denced the prominent role of the epigenetic program dys-regulation among BPDCN patients and guided our thera-peutic approach towards the use of epigenetic agents. In particular, we tested in vivo the efficacy of US Food and Drug Administration-approved epigenetic drugs which could be considered for potential repositioning in clinical
trials: two hypomethylating agents such as decitabine and 5’-azacytidine, and the histone deacetylase inhibitor romidepsin. We hypothesized that these drugs could impact on tumor progression because: i) BPDCN patients displayed potential sensitivity to hypomethylating agents, particularly to decitabine, as detected by GSEA analysis; ii) both 5’-azacytidine and decitabine are currently used for the treatment of myelodysplastic syndromes,44,45 which
are myeloid neoplasms sharing many epigenetic mutated genes with BPDCN; iii) preclinical studies on several malignancies demonstrated that the action of decitabine is synergized by romidepsin.46In the light of this, our
exper-imental design focused on epigenetic drugs with a large-scale activity, aiming to explore whether we might induce cell death by perturbation of the malignant epigenetic pro-gramme. In addition to the epigenetic drugs, we also veri-fied the efficacy of bortezomib, a proteasome inhibitor, which had previously been shown to significantly induce
in vitro and in vivo BPDCN cell death.17,18 Our experiments
revealed that the treatment with 5’-azacytidine in combi-nation with decitabine significantly inhibits disease pro-gression and extends survival (P<0.01) in a preclinical mouse model. In the past, two reports experimented the use of 5’-azacytidine in elderly BPDCN patients, though this therapeutic choice was not yet sustained by a molec-ular rationale.47,48 Here we demonstrate that 5’-azacytidine
is more effective in tumor eradication when combined with decitabine. Further studies are ongoing to elucidate the synergistic mechanisms between the two drugs.
In conclusion, we have identified the deregulation of the epigenetic program as a genetic hallmark of BPDCN and suggest a novel therapeutic approach based on the combi-nation of two hypomethylating agents, 5’-azacytidine and decitabine, to be tested in future clinical trials.
Funding
The present work was supported by the AIRC grants IG 15762 and 5x1000 10007 “Genetics-driven targeted manage-ment of lymphoid malignancies” and the Grant “Innovative approaches to the diagnosis and pharmacogenetic-based thera-pies of primary hepatic tumours, peripheral B and T-cell lym-phomas and lymphoblastic leukaemias” Strategic Programme 2010-2012 Regione Emilia Romagna - Università (all to SAP).
References
1. Chaperot L, Bendriss N, Manches O, et al. Identification of a leukemic counterpart of the plasmacytoid dendritic cells. Blood. 2001;97(10):3210-3217.
2. Swerdlow SH, Campo E, Hazzis NL, et al. Facchetti F, Jones D, Petrella T. Blastic plas-macytoid dendritic cell neoplasm. In: Swerdlow SH, Campo E, Hazzis NL, et al., eds. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008:145-147.
3. Facchetti F, Cigognetti M, Fisogni S, Rossi G, Lonardi S, Vermi W. Neoplasms derived from plasmacytoid dendritic cells. Mod Pathol. 2016;29(2):98-111.
4. Garnache-Ottou F, Feuillard J, Ferrand C, et al. Extended diagnostic criteria for plasma-cytoid dendritic cell leukaemia. Br J Haematol. 2009;145(5):624-636.
5. Pagano L, Valentini CG, Pulsoni A, et al.
Blastic plasmacytoid dendritic cell neoplasm with leukemic presentation: an Italian multi-center study. Haematologica. 2013;98(2): 239-246.
6. Roos-Weil D, Dietrich S, Boumendil A, et al. Stem cell transplantation can provide durable disease control in blastic plasmacy-toid dendritic cell neoplasm: a retrospective study from the European Group for Blood and Marrow Transplantation. Blood. 2013;121(3):440-446.
7. Pemmaraju N. Blastic plasmacytoid dendrit-ic cell neoplasm. Clin Adv Hematol Oncol. 2016;14(4):220-222.
8. Petrella T, Dalac S, Maynadie M, et al. CD4+ CD56+ cutaneous neoplasms: a distinct hematological entity? Groupe Francais d'Etude des Lymphomes Cutanes (GFELC). Am J Surg Pathol. 1999;23(2):137-146. 9. Leroux D, Mugneret F, Callanan M, et al.
CD4(+), CD56(+) DC2 acute leukemia is characterized by recurrent clonal chromoso-mal changes affecting 6 major targets: a
study of 21 cases by the Groupe Francais de Cytogenetique Hematologique. Blood. 2002;99(11):4154-4159.
10. Reichard KK, Burks EJ, Foucar MK, et al. CD4(+) CD56(+) lineage-negative malignan-cies are rare tumors of plasmacytoid dendrit-ic cells. Am J Surg Pathol. 2005;29(10):1274-1283.
11. Dijkman R, van Doorn R, Szuhai K, Willemze R, Vermeer MH, Tensen CP. Gene-expression profiling and array-based CGH classify CD4+CD56+ hematodermic neoplasm and cutaneous myelomonocytic leukemia as distinct disease entities. Blood. 2007;109(4):1720-1727.
12. Wiesner T, Obenauf AC, Cota C, Fried I, Speicher MR, Cerroni L. Alterations of the cell-cycle inhibitors p27(KIP1) and p16(INK4a) are frequent in blastic plasmacy-toid dendritic cell neoplasms. J Invest Dermatol. 2010;130(4):1152-1157. 13. Agliano A, Martin-Padura I, Marighetti P, et
novel xenograft mouse model of human blastic NK cell lymphoma/blastic plasmacy-toid dendritic cell neoplasm. Clin Cancer Res. 2011;17(19):6163-6173.
14. Jardin F, Ruminy P, Parmentier F, et al. TET2 and TP53 mutations are frequently observed in blastic plasmacytoid dendritic cell neo-plasm. Br J Haematol. 2011;153(3):413-416. 15. Lucioni M, Novara F, Fiandrino G, et al. Twenty-one cases of blastic plasmacytoid dendritic cell neoplasm: focus on biallelic locus 9p21.3 deletion. Blood. 2011;118(17):4591-4594.
16. Alayed K, Patel KP, Konoplev S, et al. TET2 mutations, myelodysplastic features, and a distinct immunoprofile characterize blastic plasmacytoid dendritic cell neoplasm in the bone marrow. Am J Hematol. 2013;88(12):1055-1061.
17. Sapienza MR, Fuligni F, Agostinelli C, et al. Molecular profiling of blastic plasmacytoid dendritic cell neoplasm reveals a unique pat-tern and suggests selective sensitivity to NF-kB pathway inhibition. Leukemia. 2014;28(8):1606-1616.
18. Philippe L, Ceroi A, Bole-Richard E, et al. Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neo-plasm. Haematologica. 2017;102(11):1861-1868.
19. Menezes J, Acquadro F, Wiseman M, et al. Exome sequencing reveals novel and recur-rent mutations with clinical impact in blastic plasmacytoid dendritic cell neoplasm. Leukemia. 2014;28(4):823-829.
20. Stenzinger A, Endris V, Pfarr N, et al. Targeted ultra-deep sequencing reveals recurrent and mutually exclusive mutations of cancer genes in blastic plasmacytoid den-dritic cell neoplasm. Oncotarget. 2014;5(15): 6404-6413.
21. Emadali A, Hoghoughi N, Duley S, et al. Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neo-plasms. Blood. 2016;127(24):3040-3053. 22. Montero J, Stephansky J, Cai T, et al. Blastic
Plasmacytoid Dendritic Cell Neoplasm Is Dependent on BCL2 and Sensitive to Venetoclax. Cancer Discov. 2017;7(2):156-164.
23. Ceroi A, Masson D, Roggy A, et al. LXR agonist treatment of blastic plasmacytoid dendritic cell neoplasm restores cholesterol efflux and triggers apoptosis. Blood. 2016;128(23):2694-2707.
24. Ceribelli M, Hou ZE, Kelly PN, et al. A Druggable TCF4- and BRD4-Dependent Transcriptional Network Sustains Malignancy in Blastic Plasmacytoid
Dendritic Cell Neoplasm. Cancer Cell. 2016;30(5):764-778.
25. Angelot-Delettre F, Roggy A, Frankel AE, et al. In vivo and in vitro sensitivity of blastic plasmacytoid dendritic cell neoplasm to SL-401, an interleukin-3 receptor targeted bio-logic agent. Haematobio-logica. 2015;100(2): 223-230.
26. Fanelli M, Amatori S, Barozzi I, et al. Pathology tissue-chromatin immunoprecipi-tation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples. Proc Natl Acad Sci U S A. 2010;107(50):21535-21540. 27. Maeda T, Murata K, Fukushima T, et al. A
novel plasmacytoid dendritic cell line, CAL-1, established from a patient with blastic natural killer cell lymphoma. Int J Hematol. 2005;81(2):148-154.
28. Ashburner M, Ball CA, Blake JA, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25(1):25-29.
29. Ward AF, Braun BS, Shannon KM. Targeting oncogenic Ras signaling in hematologic malignancies. Blood. 2012; 120(17):3397-3406.
30. Guarini A, Marinelli M, Tavolaro S, et al. ATM gene alterations in chronic lymphocyt-ic leukemia patients induce a distinct gene expression profile and predict disease pro-gression. Haematologica. 2012;97(1):47-55. 31. Simon M, Grandage VL, Linch DC, Khwaja
A. Constitutive activation of the Wnt/beta-catenin signalling pathway in acute myeloid leukaemia. Oncogene. 2005; 24(14):2410-2420.
32. Kuo YH, Zaidi SK, Gornostaeva S, Komori T, Stein GS, Castilla LH. Runx2 induces acute myeloid leukemia in cooperation with Cbfbeta-SMMHC in mice. Blood. 2009;113(14):3323-3332.
33. Togami K, Madan V, Li J, et al. Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Harbors Frequent Splicesosome Mutations That Cause Aberrant RNA Splicing Affecting Genes Critical in pDC Differentiation and Function. Blood. 2016; 128(22):738.
34. Scibetta AG, Santangelo S, Coleman J, et al. Functional analysis of the transcription repressor PLU-1/JARID1B. Mol Cell Biol. 2007;27(20):7220-7235.
35. Pal S, Vishwanath SN, Erdjument-Bromage H, Tempst P, Sif S. Human SWI/SNF-associ-ated PRMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes. Mol Cell Biol. 2004;24(21):9630-9645. 36. Missiaglia E, Donadelli M, Palmieri M,
Crnogorac-Jurcevic T, Scarpa A, Lemoine NR. Growth delay of human pancreatic can-cer cells by methylase inhibitor 5-aza-2'-deoxycytidine treatment is associated with activation of the interferon signalling path-way. Oncogene. 2005;24(1):199-211. 37. Lee KH, Park JW, Sung HS, et al. PHF2
his-tone demethylase acts as a tumor suppressor in association with p53 in cancer. Oncogene. 2015;34(22):2897-2909.
38. Subtil-Rodriguez A, Vazquez-Chavez E, Ceballos-Chavez M, et al. The chromatin remodeller CHD8 is required for E2F-depen-dent transcription activation of S-phase genes. Nucleic Acids Res. 2014;42(4):2185-2196.
39. Rotili D, Mai A. Targeting Histone Demethylases: A New Avenue for the Fight against Cancer. Genes Cancer. 2011;2(6): 663-679.
40. Jin Y, Zhou J, Xu F, et al. Targeting methyl-transferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia. J Clin Invest. 2016;126(10):3961-3980. 41. Yamane K, Tateishi K, Klose RJ, et al. PLU-1
is an H3K4 demethylase involved in tran-scriptional repression and breast cancer cell proliferation. Mol Cell. 2007;25(6):801-812. 42. Tang B, Qi G, Tang F, et al. JARID1B pro-motes metastasis and epithelial-mesenchy-mal transition via PTEN/AKT signaling in hepatocellular carcinoma cells. Oncotarget. 2015;6(14):12723-12739.
43. Zhang T, Cooper S, Brockdorff N. The inter-play of histone modifications - writers that read. EMBO Rep. 2015;16(11):1467-1481. 44. Quintas-Cardama A, Santos FP,
Garcia-Manero G. Therapy with azanucleosides for myelodysplastic syndromes. Nat Rev Clin Oncol. 2010;7(8):433-444.
45. Jabbour E, Short NJ, Montalban-Bravo G, et al. Randomized phase 2 study of low-dose decitabine vs low-dose azacitidine in lower-risk MDS and MDS/MPN. Blood. 2017;130(13):1514-1522.
46. Kalac M, Scotto L, Marchi E, et al. HDAC inhibitors and decitabine are highly syner-gistic and associated with unique gene-expression and epigenetic profiles in mod-els of DLBCL. Blood. 2011;118(20):5506-5516.
47. Laribi K, Denizon N, Ghnaya H, et al. Blastic plasmacytoid dendritic cell neoplasm: the first report of two cases treated by 5-azacy-tidine. Eur J Haematol. 2014;93(1):81-85. 48. Khwaja R, Daly A, Wong M, Mahe E,
Cerquozzi S, Owen C. Azacitidine in the treatment of blastic plasmacytoid dendritic cell neoplasm: a report of 3 cases. Leuk Lymphoma. 2016;57(11):2720-2722.
